19 January 2009. A new study led by the EPICURE Integrated Project, a European research consortium devoted to unraveling the genomics and neurobiology of epilepsy, has linked microdeletions in 15q13.3 with idiopathic generalized epilepsies (IGEs), seizure disorders that comprise up to one-third of all epilepsy cases.

Deletions in the same region have recently been associated with schizophrenia (see SRF related news story), but deletion carriers identified in the EPICURE study have no history of psychosis, and the deletion was also seen in unaffected relatives of probands with epilepsy. The wide phenotypic range reported in these studies mirrors that seen in other analyses of copy-number variations (CNVs) and presents a challenge to the common disease/common variant hypothesis that has guided most genomic research to date (see SRF related news story and Q&A with Evan Eichler and Heather Mefford [also authors on the EPICURE paper] regarding their work on CNVs in 1q21.1). The study appears in the January 11 online edition of Nature Genetics, with Thomas Sander of the University of Cologne, Germany, as corresponding author.

The 15q13–q14 region had previously been implicated in epilepsy in linkage studies (Neubauer et al., 1998; Elmslie et al., 1997; Sander et al., 2000) that proposed a pathogenic role for mutations in the CHRNA4 and CHRNA7 genes in this region, which code for the α4 and α7 subunits of the nicotinic acetylcholine receptor (see SRF related news story for a review of current thinking on cholinergic receptors and schizophrenia). More recently, Andrew Sharp, Mefford, Eichler, and colleagues at the University of Washington associated a 1.5 Mb microdeletion in the 15q13.3 region, which includes CHRNA7 and six other genes, with a syndrome characterized by mental retardation and seizures (Sharp et al., 2008).

In the new study, first author Ingo Helbig and the EPICURE team looked for the same 1.5 Mb deletion in DNA samples from 1,223 individuals with IGE and 3,699 ancestrally matched controls, and found the deletion in 12 (1 percent) of the IGE cases (including one case with an overlapping 3.8 mb deletion) but in none of the controls. The prevalence of this structural variant in the general population has been estimated at 0.02 percent, so the EPICURE study suggests that the deletion is 50 times more common in those with IGE.

However, as in earlier CNV studies, this clear conclusion is muddied by the mixed clinical picture of the probands’ relatives. In one case, the 15q13.3 deletion was de novo. In four other cases for which parental DNA was available, the deletion was inherited, yet no seizure disorders were reported in the affected parents. Three siblings who carried the deletion suffered from IGE, and a fourth had severe intellectual disability with no history of seizures. Moreover, as noted above, no psychosis was reported in any proband, and most showed none of the other phenotypes that have also been associated with the 15q13.3 deletion, which include mental retardation, autism, growth retardation, and dysmorphic features.

Surveying the recent literature, the authors write, “Taken together, the current studies reveal extensive variability in the phenotypic manifestation associated with the 15q13.3 deletion, ranging from apparently healthy individuals to severely affected individuals with a broad spectrum of neuropsychiatric disorders.... These findings...argue for a new framework for understanding complex genetic diseases.”—Pete Farley.

Before Christmas, an insightful discussion between SRF's Pete Farley and researchers Heather Mefford and Evan Eichler delved into the complex interplay between genotype (copy number variant status at 1q21.1) and phenotype (psychiatric illness, autism, mental retardation, and congenital abnormalities) (see SRF related news story). The upshot was that although deletions at this locus were statistically associated with pathologies, the severity and nature of those pathologies was extremely variable. This raised questions about whether researchers and clinicians should focus on the disease or the deletion, and what the mechanisms that determine the clinical endpoint might be. This is becoming a clear trend. Another CNV region at 16p11.2 has also been variously associated with both autism and schizophrenia. Deletions of just a single gene, CNTNAP2, as opposed to a gene cluster, have also shown this phenomenon of variable phenotype expression—deletion carriers have been diagnosed with autism, Gilles de la Tourette/obsessive compulsive disorder, schizophrenia/epilepsy, or remain entirely healthy (Bakkaloglu et al., 2008; Friedman et al., 2008; Verkerk et al., 2003; Belloso et al., 2007).

In the same vein, this new paper by Helbig and colleagues describes yet another example of a discrete copy number variant (microdeletion) that was originally linked with psychiatric phenotypes but is now also shown to give rise to idiopathic generalized epilepsy (IGE). The deletion is at 15q13.3, which encompasses the candidate neurotransmitter receptor gene, CHRNA7, among others. In fact, with a frequency of 1 percent in the IGE population and absence in controls, the deletion is the strongest genetic risk factor for this condition and is more prevalent in IGE than in either mental retardation or schizophrenia.

Although the study of CNVs has highlighted this genotype-phenotype issue, it has been observed previously in the context of the overlap of linkage hotspots between schizophrenia and bipolar disorder (Berrettini, 2003), in case-control association studies linking the same gene to multiple disorders (Chubb et al., 2008), and in the case of the Scottish family with the t(1;11) translocation disrupting DISC1, in which carrier phenotypes ranged from healthy to major depression, bipolar disorder, and schizophrenia (Blackwood et al., 2001).

So we are now faced with complex genetic disorders that really live up to their name. As such, two particular issues warrant further discussion.

The first issue is that clinicians seem to observe discrete rather than continuous disorder phenotypes. Despite the current diagnostic manuals leaving little room for diagnostic leeway, it seems that the majority of case phenotypes tend toward a limited number of outcomes such as schizophrenia, bipolar disorder, mental retardation, autism, and epilepsy. Moreover, no psychiatrist can distinguish DISC1 schizophrenia from 1q21.1 schizophrenia or NRG1 schizophrenia without recourse to genetic methodologies, suggesting that there is a positive biological drive towards the endpoint. To borrow what may be a useful analogy from physics, the system is “chaotic” (in terms of its genetic input and its effect on cellular biology) but tends toward “strange attractors” (a limited set of diagnoses) [http://en.wikipedia.org/wiki/Attractor]. Why might this be so? It may be that there are several higher order functional bottlenecks within the brain such as synaptic transmission efficiency, cortical development, astrocyte/oligodendrocyte function, hippocampal neurogenesis, higher order communication between brain regions, etc. These act to “sum” the expected environmental, genetic, and cellular complexity present within an individual and transform it into a limited set of potential outcomes—in essence, these are the strange attractors.

The next issue is how the same mutation can give rise to two (or more) different conditions. It may be useful to think of the Knudson “two-hit” hypothesis of cancer in which environment and other genetic factors act subsequent to a “deep” genetic fault (Knudson, 1971).

The CNV examples above may represent such fundamental disruptions and most probably impinge on neurodevelopmental pathways, priming the brain to be tipped over the threshold into a disease state. In fact, the t(1:11) translocation carriers present evidence for such a phenomenon as both healthy and affected carriers show abnormal P300 brain response activities suggesting this endophenotype highlights an underlying brain dysfunction (Blackwood et al., 2001).

We have to postulate that the additional genetic or environmental influences (modifiers) not only determine entry into the disease state but also dictate the final outcome. Possible candidates for modifiers of the deletions above are the remaining single copy alleles at the CNV locus—exposed recessive mutations, imprinting, or epigenetic modification could all alter expressivity and penetrance of the deletion phenotype. However, limited studies by Eichler’s group seem to discount this possibility (Mefford et al., 2008).

In any case, genomewide association and CNV studies suggest that there is plenty of scope for a sufficient burden of genetic modifiers outside the CNV region. This may also fit in with the seemingly disparate concepts of rare/familial variants exposed by linkage and common/low odds ratio variants revealed by association. Both act causally with the former potentially acting as the “first hit.”

As time progresses, we will move towards the definition of the range of phenotypes potentially resulting from each genotype and the spectrum of genotypes causing each phenotype. CNVs represent a pretty blunt tool to dissect finer relationships between genotype and phenotype, so it is to be expected that rare but penetrant point mutations that emerge from resequencing projects will be of greater use in dissecting function-phenotype links—as has been seen with the connexin gene family, for example (Rabionet et al., 2002).

In summary, it is to be hoped that the clinical and research communities are able to embrace these complexities for what they offer—a deeper understanding of these disorders, one that is intimately linked to the development and function of the brain.